The long-term goal of this project is to understand the role of membrane traffic in the cellular actions of vasopressin AVP). Newly developed anti-peptide antibodies that are specific for UT-A3 and ROMK will be used to test the hypothesis that AVP acts on these transporters to cause trafficking to the plasma membrane acutely and an increase in their abundance with long-term exposure. New extremely sensitive methods will allow quantitative confocal and electron microscopic immunolocalization studies to test this hypothesis. Additional studies will use antibodies to different sites of UT-A1 to identify the cellular location of UT-A4 and UT-A2 in the descending thin limb of short loops of Henle. This will test the hypothesis that these isoforms function as surface specific urea transporters in this segment. Trafficking will also be tested using surface biotinylation of isolated suspensions of IMCD to monitor surface expression of UT-A3 in response to AVP. Similarly, we will use biotinylation of isolated preparations of cortical collecting ducts (CCD) to test if ROMK traffics to the surface with acute AVP exposure. This preparation will also be used to test if there are changes in the abundance of ROMK in CCDs with chronic adaptation due to AVP, high K diet and aldosterone. Other studies using amphibian oocytes will test the role of PKA site 525 in trafficking of ROMK using surface biotinylation. Co-expression of the PDZ protein NHERF2 and/or ezrin will be used to examine if these proteins alter surface expression of ROMK. This will test the hypothesis that NHERF2 participates in a signaling complex important for ROMK regulation. Other studies will use colocalization of transporters with established markers to identify which distal regions express ROMK and ENaC. Studies will also test the hypothesis that adaptations can induce expression in regions that normally have little or undetectable amounts of these transporters. This will provide unique data on whether chronic adaptations to AVP, K diet and aldosterone have effects on expression of these channels that have previously been detected for water channels in the connecting tubule.